This invention relates to methods of increasing the solubility of proteins produced recombinantly. Specifically, the invention is directed to production of recombinant proteins as fusion proteins in order to increase their solubility.
Advances in molecular biology and the exploitation of recombinant DNA procedures has made possible the production of significant quantities of foreign proteins in certain host cell systems. Recombinant proteins are produced in the host cell systems by transfecting the host cells with DNA coding for the protein of interest, and then growing the transfected host cells under conditions that allow for expression of the new recombinant protein.
Prokaryotic cells have become the system of choice for expression of cloned genes encoding eukaryotic and prokaryotic polypeptides, and numerous expression systems exist for expression of gene products in bacteria. The expression of genes in E. coli has become established as a key technique in the understanding of molecular processes and E. coli expression systems have become a standard and popular method for the production and large-scale purification of exogenous proteins. Importantly, the technology has provided a source of proteins in a quantity and quality that was previously difficult, or impossible, to achieve through isolation from natural sources.
When recombinant proteins are expressed in E. coli, they frequently form insoluble protein aggregate complexes commonly referred to as xe2x80x9cinclusion bodiesxe2x80x9d. Recovery of the desired protein which is in the form of such inclusion bodies has presented a number of problems. For example, it can be difficult to isolate the inclusion bodies from other host cellular materials, and to subsequently remove inclusion body protein contaminants from the desired inclusion body protein. Additionally, the inclusion body protein is often in the form which, while identifiable as the desired protein, is not biologically active. In such instances, denaturants and detergents (e.g., guanidine hydrochloride, urea, sodium dodecylsulfate (SDS), Triton X-100) have to be used to extract the protein. The resultant solution containing the denatured protein with the individual polypeptide chains unfolded is then treated to remove the denaturant or otherwise reverse the denaturing conditions and thereby permit renaturation of the protein and xe2x80x9cfoldingxe2x80x9d of the polypeptide chains in solution to yield protein in native, biologically active form.
One strategy for avoiding the problems with refolding would be to facilitate the proper folding of recombinant protein within the cell, thus preventing the formation of inclusion bodies. The problem is that the folding processes can be quite slow and involve reactive intermediates which aggregate and compete with the slower process of folding into the native conformation. Inhibition of the competing aggregation pathway would facilitate proper folding of recombinantly expressed proteins. One way to inhibit aggregation is to enhance the solubility of recombinantly produced proteins of interest by preparing the protein of interest as a xe2x80x9cfusion proteinxe2x80x9d, i.e., fuse the protein of interest to another protein that folds readily into a highly stable conformation.
To prepare a fusion protein (also known as a xe2x80x9cchimeric proteinxe2x80x9d), the gene encoding the protein of interest can be attached to a second gene encoding a second protein, termed a xe2x80x9cfusion partnerxe2x80x9d. In this way, a single polypeptide is produced by the host cell, and the polypeptide is comprised of the protein of interest and the fusion partner. The fusion partner may be homologous (i.e., from the same species and/or strain as the host cell) or heterologous (i.e., from a species and/or strain other than that of the host cell) to the host cell. Examples of commonly used fusion partners include, inter alia, maltose binding protein (xe2x80x9cMBPxe2x80x9d), glutathione-s-transferase (xe2x80x9cGSTxe2x80x9d), hexaHistidine (xe2x80x9chexaHisxe2x80x9d) the lacZ and trpE gene products, ubiquitin, and thioredoxin. While each of these fusion partners has been demonstrated to enhance the solubility of at least one protein of interest, certain other proteins of interest do not demonstrate enhanced solubility when linked to these fusion proteins.
In certain cases, particularly where it is desirable to obtain the protein of interest in a purified form, the fusion partner and protein of interest must be separated from each other after synthesis as a single polypeptide. One means to achieve this is to provide a peptide linker between the fusion partners. This is accomplished by adding nucleic acid sequence encoding the peptide between the gene encoding the protein of interest and the gene encoding the fusion partner. Typically, this xe2x80x9clinker sequencexe2x80x9d DNA encodes an oligopeptide that contains a xe2x80x9ccleavage recognition sequencexe2x80x9d for an endopeptidase such as enterokinase, Factor Xa, caspase, or thrombin. The endopeptidase, when presented with a fusion protein containing its specific target sequence, can thus cleave the fusion protein into its two components.
For further discussions of fusion proteins see, for example, WO 95/04076, published Feb. 9, 1995; U.S. Pat. No. 5,629,172 issued May 13, 1997; WO 94/23040, published Oct. 13, 1994; Flaschel et al., Biotech Adv., 11:31-78 (1993); European patent application 207,044, published Dec. 30, 1986; U.S. Pat. No. 5,322,930, issued Jun. 21, 1994; European Patent 293,249, published Nov. 30, 1988; U.S. Pat. No. 5,654,176, issued Aug. 5, 1997; WO 95/16044, published Jun. 15, 1995; WO 94/02502, published Feb. 3, 1994; and WO 92/13955, published Aug. 20, 1992.
Uracil DNA glycosylase inhibitor (UGI) is a heat stable, acidic, low molecular weight protein expressed from the Bacillus subtilis phage, PBS1 or PBS2. The function of UGI is to inhibit the repair system that removes uracil from DNA and replaces it with thymine, allowing the phage to use uracil exclusively in its DNA. UGI has a molecular weight of 9.5 kD, a pI of 3.96, is stable to boiling, and is well-expressed in E. coli. 
It is an object of the present invention to provide new methods of enhancing the solubility of recombinant proteins produced in bacterial host cells, through utilization of UGI as a fusion partner.
In one embodiment, the present invention provides a method of increasing the solubility of a protein of interest produced in a host cell comprising expression of the protein as a fusion protein with UGI. Optionally, the protein of interest is selected from the group consisting of: extracellular domains of membrane-bound receptor proteins, cytokines and cytokine-like proteins, the catalytic domain of serine/threonine kinases, and members of the TNF family. Additionally, the host cell may be a prokaryotic cell such as a bacterial cell, and the bacterial cell may be an E. coli cell.
In yet another embodiment, the invention provides a method of increasing the solubility of a protein of interest produced in a host cell comprising expressing the protein as a fusion protein with UGI, wherein the fusion protein contains a linker peptide.